The taste bud consists of approximately 50-100 taste cells which include three morphologic types that exhibit both neuronal and epithelial properties. Based on immunocytochemical characteristics, these taste cells can be classified as type I (dark), type II (light), and type III (intermediate) (Yee et al., J Comp Neurol. 2001 Nov. 5; 440 (1):97-108; Takeda et al., J Comp Neurol. 2004 Nov. 1; 479 (1):94-102).
Studies indicate that approximately 10% of these taste cells exhibit immunoreactivity for neural cell adhesion molecule (NCAM). While NCAM-immunoreactive cells are type III cells, not all type III taste cells are immunoreactive for NCAM (Nelson and Finger 1993, J. Comp Neurol 336 (4):507-16), and NCAM expression in taste cells is dependent upon innervation by the IX nerve (Smith et al. 1994, J. Comp Neurol 347 (2):187-96). These and other data suggest that the NCAM-expressing taste cells communicate with nerves.
In contrast, key molecules required for functional responses to taste stimuli are not expressed in NCAM-immunoreactive cells. For instance, gustducin, is a key G-protein involved in taste transduction and is only present in type II cells. However, gustducin is not detected in NCAM-expressing cells. (Takeda et al. 1992, J. Electron Microsc. 41 (5):375-80; Yang et al. 2000, J. Comp Neural 425 (1):139-51).
Taste cells are believed to originate from the epithelial cell lineage with a limited average life span of 10 days, with dying cells being replaced by the basal cell population. The large majority of primary cell cultures of taste cells are reported to last a few days at best, e.g. 3 to 5 days maximum (compare, for example, for mouse taste cells a method modified from the isolation of mammalian central neurons by Spielman et al. 1989, Brain Research 503: 326-29; and for rat taste cells Kishi et al. 2001, Neuroscience 106 (1): 217-25, and Stone et al. 2002, Chem. Senses 27: 779-87).
To be able to detect a given tastant, taste cells typically need both the relevant taste receptor for the tastant (e.g., bitter, sweet, umami) and the molecules necessary for taste transduction. Taste receptors may include one or more of T2Rs and/or one or more of T1Rs. Signal transduction molecules may include gustducin and phospholipase C. Cells that express at least one taste receptor and are able to respond to at least one taste stimulus/tastant are referred to herein as “taste receptor cells.” Taste cells thus comprise taste receptor cells among other cell types.
To date, there is no cell culture model for taste receptor cells and any in vitro research has had to rely on primary cell cultures of taste cells that are maintained for a limited time.
Ookura et al. (2002, In Vitro Cell. Dev. Biol.-Animal 38: 365-72) describe a particular type of cells isolated from mouse taste epithelium that have been sorted based on their integrin β1 marker and that express the NCAM marker. Thus, this integrin-positive continuous mouse cell culture docs not generate cells similar to those responsible for the primary detection of taste stimuli.
Ruiz et al. (2001, Chem. Senses 26 (7):861-73) report the maintenance of primary cell cultures derived from taste buds for up to 14 days, provided the cells are kept at room temperature, which is believed to slow down various cellular processes. Notably, cells kept at 37° C. could be maintained only for a few days, as had been previously reported. The cells kept at room temperature are reported to start dying at around day 10, which corresponds to the expected average life span of taste cells. A protocol published by Ruiz et al. 1995 (“Tissue Culture of Rat Taste Buds”, Eds Spielman A I, Brand J G Experimental Cell Biology of Taste and Olfaction, CRC Press, 1995) discloses a similar method at room temperature that mentions a culture duration of up to 18 days.